Because of concern about the safety of immunoglobulins with respect to transmission of hepatitis C, we examined the partitioning of hepatitis C virus (HCV) during alcohol fractionation of a plasma pool prepared exclusively from anti-HCV reactive donations. Quantitation of HCV RNA was accomplished by nested polymerase chain reaction (PCR) at limiting dilution. One PCR unit was arbitrarily defined as the minimum amount of HCV RNA from which an amplified product could be detected. The sensitivity of the PCR assay for HCV RNA was determined by performing limiting dilution analysis on a sample of infectious plasma (H strain) known to contain 106.6 chimpanzee infectious doses/ml. In our assay, this sample contained 1.4 x 106 PCR units of HCV RNA/ml. Thus, the PCR assay has a sensitivity comparable to the chimpanzee model. When 3,073 plasma donations from otherwise acceptable donors were tested for anti-HCV, 186 were repeatedly reactive and 2,887 were negative. A pool prepared from the anti-HCV reactive donations contained 1.4 x 105 PCR units of HCV RNA/ml, whereas a pool prepared from the negative donations contained 1.6 x 103 PCR units/ml. It can be calculated that a pool comprised of all 3,073 units would contain 1.0 x 104 PCR units/ ml. Thus, in this instance, anti-HCV screening decreased the viral load of the plasma pool by a factor of 6. A 100-ml sample of the anti-HCV reactive pool was fractionated to immune globulin by the Cohn-Oncley procedure, and samples of the various fractions were analyzed for HCV RNA. Most of the HCV RNA was found in cryoprecipitate and in Cohn fractions I and III, but it was also detected in fraction II, used for immunoglobulin G preparations. A 3.4% solution of IgG prepared from this fraction II contained 30 PCR units/ml. The fractionation process leading to immune globulin resulted in an overall reduction in HCV RNA by a factor of 4.7 x 104. Although the presence of HCV RNA in the final product does not necessarily imply the presence of virus, this work suggests that the safety of immune globulins with respect to HCV transmission is not due solely to partitioning of HCV away from the immunoglobulin fraction.